The advent of tissue engineering and biomedical techniques has significantly advanced the development of three-dimensional (3D) cell culture systems, particularly tumor organoids. These self-assembled 3D cell clusters closely replicate the histopathological, genetic, and phenotypic characteristics of primary tissues, making them invaluable tools in cancer research and drug screening.

This review addresses the challenges in developing in vitro models that accurately reflect tumor heterogeneity and explores the application of tumor organoids in cancer research, with a specific focus on the screening of natural products for antitumor therapies.

This review synthesizes information from major databases, including Chemical Abstracts, Medicinal and Aromatic Plants Abstracts, ScienceDirect, Google Scholar, Scopus, PubMed and Springer Link. Publications were selected without date restrictions, using terms such as 'organoid', 'natural product', 'pharmacological', 'extract', 'nanomaterial' and 'traditional uses'. Articles related to agriculture, ecology, synthetic work or published in languages other than English were excluded.

The review identifies key challenges related to the efficiency and variability of organoid generation and discusses ongoing efforts to enhance their predictive capabilities in drug screening and personalized medicine. Recent studies utilizing patient-derived organoid models for natural compound screening are highlighted, demonstrating the potential of these models in developing new classes of anticancer agents. The integration of natural products with patient-derived organoid models presents a promising approach for discovering novel anticancer compounds and elucidating their mechanisms of action.

Single-cell RNA sequencing (scRNA-seq) has transformed the study of retinal development and diseases by enabling a detailed analysis of cellular diversity within retinal organoids (ROs). ROs generated from pluripotent stem cells mimic the essential characteristics of the human retina and provide a valuable in vitro model for investigating retinal development, cell interactions, and disease mechanisms. This review summarizes the application of scRNA-seq on RO research, emphasizing its capacity to identify distinct cell populations, uncover developmental trajectories, and reveal the molecular signatures of retinal diseases. scRNA-seq provides new insights into retinal neurogenesis, cellular diversity, and the pathophysiology of retinal degenerative diseases. This technology has enabled the identification of novel biomarkers and potential therapeutic targets. Integrating scRNA-seq with other technologies, such as spatial transcriptomics and CRISPR-based screening, can further deepen our understanding of retinal biology and improve treatment strategies.

Regeneration of small intestinal mucosal tissue could offer a promising strategy for Crohn's disease patients suffering from chronic inflammatory damage. Here, we aimed to develop hydrogels that mirror the villi and crypts of the small intestine and exhibit a physiological stiffness of G' ~ 1.52 kPa. For this purpose, we developed gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-, and gelatin-methacryloyl-norbornene (gel-MA-NB)-based biomaterial inks to fabricate 3D hydrogels ("villi only" versus "crypts and villi") with digital light processing (DLP) and co-cultured Caco-2/HT29-MTX cells. Gel-MA-AEMA was selected for its higher amount of methacrylates which was hypothesized to provide superior photo-crosslinking kinetics and hence superior DLP fabrication potential while gel-MA-NB was evaluated for its selective functionalization potential with thiolated bioactive compounds following DLP processing, resulting from its incorporated NB moieties which remain unreacted during the DLP process. Both gel-MA-AEMA-, and gel-MA-NB-based hydrogels exhibited a physiologically relevant stiffness, but only the gel-MA-AEMA-based biomaterial ink could be successfully utilized for printing hydrogels encompassing villi and crypts. Paracellular permeability of small sized marker molecules in combination with transepithelial electrical resistance measurements showed the formation of a functional barrier over time on all hydrogel constructs. Transmission electron microscopy and enterocyte differentiation marker genes' expression levels revealed the superior differentiation of Caco-2 on the 3D constructs compared to 2D hydrogel sheets. In summary, while both hydrogels enhanced functional barrier formation and enterocyte differentiation, gel-MA-AEMA proved more conducive to DLP compared to gel-MA-NB. Furthermore, our study underscored the benefits of cultivating intestinal cells on soft 3D constructs, enhancing cell barrier properties and differentiation, thus providing added value over traditional 2D supports.

Cancer stem cells (CSCs) contribute to chemotherapy resistance and poor prognosis, posing significant challenges in the treatment of oral squamous cell carcinoma. The extracellular matrix (ECM)-constructed microenvironment remodels the niche of CSCs. Yet mechanisms by which biophysical properties of ECM relate to CSCs remain undefined. Here, our findings link ECM mechanical stimuli to CSCs phenotype transition, and propose that ECM stiffening mechanoactivates tumor cells to dedifferentiate and acquire CD44+ stem cell-like characteristics through noncanonical mechanotransduction. ITGB1 senses and transduces biomechanical signals, while FERMT1 acts as an intracellular mechanotransduction downstream, activating CSCs. Mechanistically, FERMT1 promotes the proteasomal degradation of CK1α by E3 ubiquitin ligase MIB1, thereby triggering Wnt signaling pathway. Combining targeted ECM softening with mechanotransduction inhibition strategy significantly attenuates tumor stemness and chemoresistance in vivo. Therefore, our findings highlight the role of ECM in regulating CSCs via biomechanical-dependent manner, suggesting the ECM/ITGB1/FERMT1/Wnt axis as a promising therapeutic target for CSCs therapy.

The mechanical properties of the hydrogel play a pivotal role in governing the formation and development of 3D organoids in vitro. However, commonly employed natural hydrogels, such as Matrigel and other extracellular matrix (ECM)-derived products, are characterized by ill-defined and complex compositions, resulting in non-tunable mechanical properties. This limitation poses challenges in controlling organoids' developmental trajectory and 3D morphology. Although numerous synthetic hydrogels with well-defined chemical structures have recently been adopted to study organoids by modulating stiffness, advanced research emphasizes the importance of dynamic mechanical cues, such as dynamic stiffness softening and dynamic viscoelasticity, for optimal organoid derivation. These cues are essential for mimicking the dynamic physiological states of organoids during their growth. Despite their potential, the concept of dynamic hydrogels is often used interchangeably, and a systematic review is lacking to clarify this ambiguity. Furthermore, the mechanisms through which dynamic mechanical cues regulate organoid formation have not been thoroughly reported. This review endeavors to summarize and categorize dynamic hydrogels and reveal the effects of dynamic mechanics on organoid derivation. Additionally, the prospects of dynamic hydrogels in organoid derivation are deliberated to promote a more rational design of synthetic hydrogels, guiding organoid derivation and propelling organoid technology in biomedicine.

Advances in organoid models, as ex vivo mini-organs, and the development of screening imaging technologies have continuously driven each other forward. A complete understanding of organoids requires detailed insights into the intertwined intraorganoid and extraorganoid activities and how they change across time and space. This study introduces a multiplexed imaging platform that integrates label-free nanoplasmonic biosensing with fluorescence microscopy to offer simultaneous monitoring of dynamics occurring within and around arrays of single spheroids with spatiotemporal resolution. The label-free module employs nanoplasmonic biosensors with extraordinary optical transmission to track biomolecular secretions into the surroundings, while concurrent fluorescence imaging enables structural analysis and viability assessment. To perform multiparametric interrogation of the data from different channels over extended periods, a deep-learning-augmented image analysis is incorporated. The platform is applied to tumor spheroids to investigate vascular endothelial growth factor A secretion alongside morphometric changes and viability, showcasing its ability to capture variations in secretion and growth dynamics between untreated and drug-treated groups. This integrated approach advances comprehensive insights into organoid models and can complement existing technologies to accelerate discoveries in disease modeling and drug development.

Ovarian cancer (OC) poses significant treatment challenges due to late-stage diagnosis and a complex tumor microenvironment contributing to therapy resistance. We optimized a U-CUP perfusion-based bioreactor method to culture patient-derived primary and metastatic OC specimens, demonstrating that perfusion better preserves cancer cell viability and proliferation, both when fresh and slow-frozen tissues were used. Perfused cultures maintained key microenvironment components, including cancer-associated fibroblasts, endothelial and immune cells. Genetic analysis confirmed the retention in culture of tumor-specific driver mutations. We hence challenged ad hoc generated cisplatin-sensitive and resistant OC cells with cisplatin during growth in U-CUP, validating our system for the testing of drug response. Finally, treatment of slow-frozen OC tissues with carboplatin/paclitaxel revealed different degrees of response to treatment, as indicated by variations in tumor necrosis and number of residual PAX8+ cells, providing the bases for the prompt evaluation of OC standard chemotherapy efficacy in our ex vivo system.

Intestinal organoids are valuable tools across different disciplines, from a clinical aspect to the biomedical research, providing a unique perspective on the complexity of the gastrointestinal system. They are alternatives to common cell lines as they can offer insights into architectural functionality and reduce the use of animal models. A deeper understanding of their organoid characteristics is required to harness their full potential. Despite their beneficial uses and multiple advantages, organoids have limitations that remain unaddressed. This review aims to elucidate the principal limitations of intestinal organoids, investigate structural defects such as the deficiency in a vascularized and lymphatic system, and absence of the microbiome, restrictions in mimicking the physiological gut model, including the lack of an acid-neutralizing system or a shortage of digestive enzymes, and the difficulties in their long-term maintenance and polarity accessibility. Development of innovative techniques to address these limitations will lead to improve in vivo recapitulation and pioneering further advancements in this field.

The advent of intestinal organoids, three-dimensional structures derived from stem cells, has significantly advanced the field of biology by providing robust in vitro models that closely mimic the architecture and functionality of the human intestine. These organoids, generated from induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or adult stem cells, possess remarkable capabilities for self-renewal, differentiation into diverse intestinal cell types, and functional recapitulation of physiological processes, including nutrient absorption, epithelial barrier integrity, and host-microbe interactions. The utility of intestinal organoids has been extensively demonstrated in disease modeling, drug screening, and personalized medicine. Notable examples include iPSC-derived organoids, which have been effectively employed to model enteric infections, and ESC-derived organoids, which have provided critical insights into fetal intestinal development. Patient-derived organoids have emerged as powerful tools for investigating personalized therapeutics and regenerative interventions for conditions such as inflammatory bowel disease (IBD), cystic fibrosis, and colorectal cancer. Preclinical studies involving transplantation of human intestinal organoids into murine models have shown promising outcomes, including functional integration, epithelial restoration, and immune system interactions. Despite these advancements, several challenges persist, particularly in achieving reproducibility, scalability, and maturation of organoids, which hinder their widespread clinical translation. Addressing these limitations requires the establishment of standardized protocols for organoid generation, culture, storage, and analysis to ensure reproducibility and comparability of findings across studies. Nevertheless, intestinal organoids hold immense promise for transforming our understanding of gastrointestinal pathophysiology, enhancing drug development pipelines, and advancing personalized medicine. By bridging the gap between preclinical research and clinical applications, these organoids represent a paradigm shift in the exploration of novel therapeutic strategies and the investigation of gut-associated diseases.

Cardioids are 3D self-organized heart organoids directly derived from induced pluripotent stem cells (hiPSCs) aggregates. The growth and culture of cardioids is either conducted in suspension culture or heavily relies on Matrigel encapsulation. Despite the significant advancements in cardioid technology, reproducibility remains a major challenge, limiting their widespread use in both basic research and translational applications. Here, for the first time, we employed synthetic, matrix metalloproteinase (MMP)-degradable polyethylene glycol (PEG)-based hydrogels to define the effect of mechanical and biochemical cues on cardioid development. Successful cardiac differentiation is demonstrated in all the hydrogel conditions, while cardioid cultured in optimized PEG hydrogel (3 wt.% PEG-2mM RGD) underwent similar morphological development and comparable tissue functions to those cultured in Matrigel. Matrix stiffness and cell adhesion motif play a critical role in cardioid development, nascent chamber formation, contractile physiology, and endothelial cell gene enrichment. More importantly, synthetic hydrogel improved the reproducibility in cardioid properties compared to traditional suspension culture and Matrigel encapsulation. Therefore, PEG-based hydrogel has the potential to be used as an alternative to Matrigel for human cardioid culture in a variety of clinical applications including cell therapy and tissue engineering.

Nonalcoholic fatty liver disease (NAFLD) is characterized by increased lipid accumulation and excessive deposition of extracellular matrix (ECM) that results in tissue stiffening. The potential interplay between matrix stiffness and hepatocyte lipid accumulation during NAFLD has not been established. Here, an in vitro NAFLD model is developed using chemically defined, engineered hydrogels and human induced pluripotent stem cell-derived hepatic organoids (HOs). Specifically, dynamic covalent chemistry crosslinking, along with transient small molecule competitors, are used to create dynamic stiffening hydrogels that enable the reproducible culture of HOs. Within matrices that mimic the stiffness of healthy to diseased tissue (≈1-6 kPa), lipid droplet accumulation in HOs is triggered by exposure to an NAFLD-associated free fatty acid. These NAFLD model suggests that higher stiffness microenvironments result in increased hepatic lipid droplet accumulation, increased expression of fibrosis markers, and increased metabolic dysregulation. By targeting the ROCK mechanosignaling pathway, the synergy between matrix stiffness and lipid droplet accumulation is disrupted. The in vitro model of NAFLD has the potential to understand the role of mechanosignaling in disease progression and identify new pathways for therapeutic intervention.

Cardiovascular diseases (CVD) are the primary cause of death and disability around the world. Over the past decades, several conventional model systems based on two-dimensional (3D) monolayer cultures or experimental animals have been adopted to dissect and understand heart diseases in order to develop treatment modalities. However, traditional models exhibit several limitations in recapitulating human-specific key physiological and pathological characteristics, which highlights the necessity of developing physiologically relevant models. In recent years, tissue engineering approaches have been extensively employed to generate revolutionary three-dimensional (3D) cardiac models. In particular, the combined use of various bioengineering strategies and cellular reprogramming approaches has facilitated the development of various models. This review presents an overview of different approaches (bioprinting, scaffolding, and electrospinning) for creating bioengineered cardiac tissue models. Next, a broad survey of recent research related to the modeling of various cardiac diseases is presented. Finally, current challenges and future directions are proposed to foster further developments in the field of cardiac tissue engineering.

Colorectal cancer (CRC) studies in vitro have been conducted almost exclusively on 2D cell monolayers or suspension spheroid cultures. Though these platforms have shed light on many important aspects of CRC biology, they fail to recapitulate essential cell-matrix interactions that often define in vivo function. Toward filling this knowledge gap, synthetic hydrogel biomaterials with user-programmable matrix mechanics and biochemistry have gained popularity for culturing cells in a more physiologically relevant 3D context. Here, using a poly(ethylene glycol)-based hydrogel model, we systematically assess the role of matrix stiffness and fibronectin-derived RGDS adhesive peptide presentation on CRC colony morphology and proliferation. Highlighting platform generalizability, we demonstrate that these hydrogels can support the viability and promote spontaneous spheroid or multicellular aggregate formation of six CRC cell lines that are commonly utilized in biomedical research. These gels are engineered to be fully degradable via a "biologically invisible" sortase-mediated reaction, enabling the triggered recovery of single cells and spheroids for downstream analysis. Using these platforms, we establish that substrate mechanics play a significant role in colony growth: soft conditions (∼300 Pa) encourage robust colony formation, whereas stiffer (∼2 kPa) gels severely restrict growth. Tuning the RGDS concentration did not affect the colony morphology. Additionally, we observe that epidermal growth factor receptor (EGFR) signaling in Caco-2 cells is influenced by adhesion ligand identity─whether the adhesion peptide was derived from collagen type I (DGEA) or fibronectin (RGDS)─with DGEA yielding a marked decrease in the level of downstream protein kinase phosphorylation. Taken together, this study introduces a versatile method to culture and probe CRC cell-matrix interactions within engineered 3D biomaterials.

Precision oncology is becoming a mainstay in the standard of care for cancer patients. Recent technological advancements have significantly lowered the cost of various tumor profiling approaches, broadening the reach of molecular diagnostics and improving patient access to precision oncology. In parallel, drug development and discovery pipelines continue to evolve, driving targeted therapeutic options forward. Yet, not all patients harboring actionable molecular alterations respond to these interventions, and existing therapies do not cover the entire spectrum of potential molecular targets. In this review, we examine the current suite of omics technologies employed in clinical settings and underscore their roles in deepening our understanding of tumor biology and optimizing patient stratification for targeted treatments. We also highlight relevant precision oncology trials and share our own experiences using multi-omics data within a molecular tumor board framework. Finally, we discuss areas for future exploration aimed at propelling precision oncology to new heights.

Tumor organoid models have emerged as valuable 3D in vitro systems to study cancer behavior in a physiologically relevant environment. The composition and architecture of the extracellular matrix (ECM) play critical roles in tumor organoid culture by influencing the tumor microenvironment and tumor behavior. Traditional matrices such as Matrigel and collagen, have been widely used, but their batch-to-batch variability and limited tunability hinder their reproducibility and broader applications. To address these challenges, researchers have turned to synthetic/engineered matrices and biopolymer-based matrices, which offer precise tunability, reproducibility, and chemically defined compositions. However, these matrices also present challenges of their own. In this review, we explore the significance of ECMs in tumor organoid culture, discuss the limitations of commonly used matrices, and highlight recent advancements in engineered/synthetic matrices for improved tumor organoid modeling.

Primary central nervous system lymphoma (PCNSL) exhibits substantial intratumoural and intertumoural heterogeneity, complicating the development of effective treatment methods. Existing in vitro models fail to simulate the cellular and mutational diversity of native tumours and require prolonged generation times. Therefore, we developed a culture method for patient-derived PCNSL organoids (CLOs) and evaluated the organoids through extensive molecular characterisation, histopathological analysis, single-nucleus RNA sequencing, bulk RNA sequencing and whole-exome sequencing. These CLOs accurately mimicked the histological attributes, gene expression landscapes and mutational profiles of their original tumours. Single-nucleus RNA sequencing also revealed that CLOs maintained cell-type heterogeneity and the molecular signatures of their original tumours. CLOs were generated within 2 weeks, demonstrating rapid development and reliability. Therapeutic profiling was performed on three selected CLOs treated with four standard drugs. The CLOs exhibited specific sensitivity to methotrexate, and resistance to dexamethasone, ibrutinib and rituximab, suggesting that CLOs may be valuable tools for reflecting drug sensitivities. Taken together, these results emphasise that CLOs effectively emulate the key characteristics of PCNSL, increasing the understanding of the genetic landscape of this complex disease. CLOs provide a rapid and reliable platform for exploring individualised treatment strategies, potentially accelerating the transition of research findings to clinical practice.

Heterogeneity and the absence of a tumor microenvironment (TME) in traditional patient-derived organoid (PDO) cultures limit their effectiveness for clinical use. Here, Embedded Bioprinting-enabled Arrayed PDOs (Eba-PDOs) featuring uniformly arrayed colorectal cancer (CRC) PDOs within a recreated TME is presented. This model faithfully reproduces critical TME attributes, including elevated matrix stiffness (≈7.5 kPa) and hypoxic conditions found in CRC. Transcriptomic and immunofluorescence microscopy analysis reveal that Eba-PDOs more accurately represent actual tissues compared to traditional PDOs. Furthermore, Eba-PDO effectively capture the variability of CEACAM5 expression-a critical CRC marker-across different patients, correlating with patient classification and differential responses to 5-fluorouracil treatment. This method achieves an uniform size and shape within PDOs from the same patient while preserving distinct morphological features among those from different individuals. These features of Eba-PDO enable the efficient development of a label-free, morphology-based predictive model using supervised learning, enhancing its suitability for clinical applications. Thus, this approach to PDO bioprinting is a promising tool for generating personalized tumor models and advancing precision medicine.

The cellular organization within organoid models is important to regulate tissue specific function, yet few engineering approaches can control or direct cellular organization. Here, a photodegradable hydrogel is used to create softened regions that direct crypt formation within intestinal organoids, where the dimensions of the photosoftened regions generate predictable and defined crypt architectures. Guided by in vivo metrics of crypt morphology, this photopatterning method is used to control the width and length of in vitro organoid crypts, which ultimately defines the curvature of the epithelium. By tracking expression of differentiated Paneth cell markers in real-time, we show that epithelial curvature directs the localization of Paneth cells within engineered crypts, providing user-directed control over organoid functionality. We anticipate that our improved control over organoid architecture and thus Paneth cell localization will lead to more consistent in vitro organoid models for both mechanistic studies and translational applications.

Diabetic Kidney Disease (DKD) represents a significant global health burden and is recognised as the leading cause of end-stage renal disease. Kidney organoids derived from human induced Pluripotent Stem Cells (hiPSCs) have the potential to transform how we model renal disease and may provide personalised replacement tissues for patients with renal failure. However, kidney organoids remain poorly reproducible, and are structurally and functionally immature. Three-dimensional cultures that more appropriately mimic the complexity of the in vivo microenvironment are required to improve organoid maturation and structural authenticity. Here, we describe the application of semi-synthetic Gelatin Methacryloyl (GelMA) hydrogels as extracellular support matrices for the differentiation of hiPSC-derived kidney organoids. Hydrogels of defined mechanical strengths were generated by varying the concentration of GelMA solution in the presence of low concentration photo-initiator. After confirming a high level of mechanical stability of the hydrogels over extended culture periods, their effect on kidney organoid maturation was investigated. Organoids differentiated within GelMA hydrogels generated typical renal cell types including podocytes, tubular epithelia, renal interstitial cells, and some nascent vascularisation. Interestingly, kidney organoids derived within hydrogels that closely approximate the stiffness of the adult human kidney (∼5000-10,000 Pa) demonstrated improved podocyte maturation and were shown to upregulate renal vesicle-associated genes at an earlier stage following encapsulation when compared to organoids derived within softer hydrogels (∼400 Pa). A model of TGFβ-induced injury was also developed to investigate the influence of the mechanical environment in propagating early, fibrotic-like features of DKD within organoids. Growth within the softer matrix was shown to reduce pSMAD3 expression following TGFβ1 treatment, and accordingly ameliorate the expression of the myofibroblast marker α-Smooth Muscle Actin (α-SMA). This work demonstrates the suitability of GelMA hydrogels as mechanically-stable, highly-tuneable, batch-to-batch reproducible three-dimensional supports for hiPSC-derived kidney organoid growth and differentiation, and further substantiates the role of the biophysical environment in guiding processes of cell fate determination and organoid maturation.

With the rapid advancement of biomaterials and tissue engineering technologies, organoid research and its applications have made significant strides. Organoids are increasingly utilized in pharmacology, regenerative medicine, and precision clinical medicine. Current trends in organoid research are moving toward multifunctional composite three-dimensional cultivation and dynamic cultivation strategies. Key technologies driving this evolution, including 3D printing and microfluidics, continue to impact new areas of discovery and clinical relevance. This review provides a systematic overview of these emerging trends, discussing the strengths and limitations of these critical technologies and offering insight and research directions for professionals working in the organoid field.

Culture platforms that closely mimic the spatial architecture, cellular diversity, and extracellular matrix composition of native tissues can serve as invaluable tools for a range of scientific discovery and biomedical applications. Organoids have emerged as a promising alternative to both traditional 2D cell culture and animal models, offering a physiologically relevant 3D culture system for studying human cell biology. Organoids provide a manipulable platform to investigate organ development and function as well as to model patient-specific phenotypes. This mini review examines various methods used for culturing organoids to model normal and disease conditions in gastrointestinal tissues. We focus on how the matrix composition and media formulations can impact cell signaling, altering the baseline cellular phenotypes as well as response to perturbations. We discuss future directions for optimizing organoid culture conditions to improve basic and translational potential.

Oral cancer poses significant health risks with an increasing incidence annually. Despite advancements in treatment methods, their efficacy is frequently constrained by cancer heterogeneity and drug resistance, leading to minimal improvement in the 5-year survival rate. Therefore, there is a critical need for new treatment methods leaded by representative preclinical research models. Compared to other models, organoids can more precisely simulate the tissue structure, genetic characteristics, and tumor microenvironment (TME) of in vivo tumors, exhibiting high tumor specificity. This makes organoid technology a valuable tool in investigating tumor development, mechanisms of metastasis, drug screening, prediction of clinical responses, and personalized patient treatment. Moreover, integrating organoid technology with other biotechnologies could expand its applications in tissue regeneration. Although organoid technology is increasingly utilized in oral cancer research, a systematic review in this field is absent. This paper is to bridge the gap by reviewing the development and current status of organoid research, highlighting its applications, future prospects, and challenges in oral cancer. It aims to provide novel insights into the role of organoids in precision treatment and regenerative medicine for oral cancer.

Organoids are 3D artificial miniature organs composed of a cluster of self-renewing and self-organizing cells in vitro, which mimic the functions of real organs. Nanotechnologies, including the preparation of nanomaterials and the fabrication of micro/nanostructures, have been proven to promote cell proliferation, guide cell differentiation, and regulate cell self-organization, showing great promise in engineering organoids. In this Perspective, different types of nanocomposite hydrogels for organoid culture are summarized, the effects of micro/nanostructures on organoid growth and development are discussed, and 3D bioprinting technologies for constructing organoid models are introduced.

The ubiquitous presence of per- and polyfluoroalkyl substances (PFAS) in the environment has garnered global public concern. Epidemiological studies have proved that exposure to PFAS is associated with human health risks. Although evidence demonstrated the toxic mechanisms of PFAS based on animal models and traditional cell cultures, their limitations in inter-species differences and lack of human-relevant microenvironments hinder the understanding of health risks from PFAS exposure. There is an increasing necessity to explore alternative methodologies that can effectively evaluate human health risks. Human organoids derived from stem cells accurately mimic the sophisticated and multicellular structures of native human organs, providing promising models for toxicology research. Advanced organoids combined with innovative technologies are expected to improve understanding of the breadth and depth of PFAS toxicity.

The high failure rates in clinical drug development based on animal models highlight the urgent need for more representative human models in biomedical research. In response to this demand, organoids and organ chips were integrated for greater physiological relevance and dynamic, controlled experimental conditions. This innovative platform-the organoids-on-a-chip technology-shows great promise in disease modeling, drug discovery, and personalized medicine, attracting interest from researchers, clinicians, regulatory authorities, and industry stakeholders. This review traces the evolution from organoids to organoids-on-a-chip, driven by the necessity for advanced biological models. We summarize the applications of organoids-on-a-chip in simulating physiological and pathological phenotypes and therapeutic evaluation of this technology. This section highlights how integrating technologies from organ chips, such as microfluidic systems, mechanical stimulation, and sensor integration, optimizes organoid cell types, spatial structure, and physiological functions, thereby expanding their biomedical applications. We conclude by addressing the current challenges in the development of organoids-on-a-chip and offering insights into the prospects. The advancement of organoids-on-a-chip is poised to enhance fidelity, standardization, and scalability. Furthermore, the integration of cutting-edge technologies and interdisciplinary collaborations will be crucial for the progression of organoids-on-a-chip technology.

For several decades, research has studied the influence of the extracellular matrix (ECM) mechanical properties in cell response, primarily emphasising its elasticity as the main determinant of cell and tissue behaviour. However, the ECM is not purely elastic; it is viscoelastic. ECM viscoelasticity has now emerged as a major regulator of collective cell dynamics. This review highlights recent findings on the role of ECM viscoelasticity in development and pathology.

Mechanobiology studies how mechanical forces influence biological processes at different scales, both in homeostasis and in pathology. Organoids, 3D structures derived from stem cells, are particularly relevant tools for modeling tissues and organs in vitro. They currently constitute one of the most suitable models for mechanobiology studies. This review provides an overview of existing or applicable approaches to organoids for mechanical studies. We first present the different types of culture supports, including hydrogels and organ-on-chip. We then discuss advanced imaging techniques, particularly suitable for studying the physical properties of cells, allowing the visualization of mechanical forces and cellular responses. We also describe the approaches and tools available to observe the organoids by microscopy. Finally, we present analytical methods, including computational models and biophysical measurement approaches, which facilitate the quantification of mechanical interactions. This review aims to provide the most comprehensive overview possible of the methods, instrumentations, and tools available to conduct a mechanobiological study on organoids.

Gut organoids are 3D cellular structures derived from adult or pluripotent stem cells, capable of closely replicating the physiological properties of the gut. These organoids serve as powerful tools for studying gut development and modeling the pathogenesis of intestinal diseases. This review provides an in-depth exploration of technological advancements and applications of gut organoids, with a focus on their construction methods. Additionally, the potential applications of gut organoids in disease modeling, microenvironmental simulation, and personalized medicine are summarized. This review aims to offer perspectives and directions for understanding the mechanisms of intestinal health and disease as well as for developing innovative therapeutic strategies.

The dynamic nature of cellular microenvironments, regulated by the viscoelasticity and enzymatic cleavage of the extracellular matrix, remains challenging to emulate in engineered synthetic biomaterials. To address this, a novel platform of cell-instructive hydrogels is introduced, composed of two concurrently forming interpenetrating polymer networks (IPNs). These IPNs consist of the same basic building blocks - four-armed poly(ethylene glycol) and the sulfated glycosaminoglycan (sGAG) heparin - are cross-linked through either chemical or physical interactions, allowing for precise and selective tuning of the hydrogel's stiffness, viscoelasticity, and proteolytic cleavability. The studies of the individual and combined effects of these parameters on stem cell behavior revealed that human mesenchymal stem cells exhibited increased spreading and Yes-associated protein transcriptional activity in more viscoelastic and cleavable sGAG-IPN hydrogels. Furthermore, human induced pluripotent stem cell (iPSC) cysts displayed enhanced lumen formation, growth, and pluripotency maintenance when cultured in sGAG-IPN hydrogels with higher viscoelasticity. Inhibition studies emphasized the pivotal roles of actin dynamics and matrix metalloproteinase activity in iPSC cyst morphology, which varied with the viscoelastic properties of the hydrogels. Thus, the introduced sGAG-IPN hydrogel platform offers a powerful methodology for exogenous stem cell fate control.

Precision medicine is a personalized medical model based on the individual's genome, phenotype, and lifestyle that provides tailored treatment plans for patients. In this context, tumor organoids, a 3-dimensional preclinical model based on patient-derived tumor cell self-organization, combined with digital analysis methods, such as high-throughput sequencing and image processing technology, can be used to analyze the genome, transcriptome, and cellular heterogeneity of tumors, so as to accurately track and assess the growth process, genetic characteristics, and drug responsiveness of tumor organoids, thereby facilitating the implementation of precision medicine. This interdisciplinary approach is expected to promote the innovation of cancer diagnosis and enhance personalized treatment. In this review, the characteristics and culture methods of tumor organoids are summarized, and the application of multi-omics, such as bioinformatics and artificial intelligence, and the digital methods of organoids in precision medicine research are discussed. Finally, this review explores the main causes and potential solutions for the bottleneck in the clinical translation of digital tumor organoids, proposes the prospects of multidisciplinary cooperation and clinical transformation to narrow the gap between laboratory and clinical settings, and provides references for research and development in this field.

Biomimetic gut models show promise for enhancing our understanding of intestinal disorder pathogenesis and accelerating therapeutic strategy development. Currentin vitromodels predominantly comprise traditional static cell culture and animal models. Static cell culture lacks the precise control of the complex microenvironment governing human intestinal function. Animal models provide greater microenvironment complexity but fail to accurately replicate human physiological conditions due to interspecies differences. As the available models do not accurately reflect the microphysiological environment and functions of the human intestine, their applications are limited. An optimal approach to intestinal modeling is yet to be developed, but the field will probably benefit from advances in biofabrication techniques. This review highlights biofabrication strategies for constructing biomimetic intestinal models and research approaches for simulating key intestinal physiological features. We also discuss potential biomedical applications of these models and provide an outlook on multi-scale intestinal modeling.

Host-microbe interactions in the intestine play a significant role in health and disease. Novel scaffolds for host cells, capable of potentially supporting these intricate interactions, are necessary to improve our current systems for mimicking host-microbiota interplay in vitro/ex vivo. In this research paper, we study the application of gelatin methacrylamide (GelMA) as scaffold material for intestinal epithelial cells in terms of permeability, mechanical strength, and biocompatibility. We investigated whether the degree of substitution (DS) of GelMA influences the permeability and found that both high and low DS GelMA show sufficient permeability of biorelevant transport markers. Additionally, we researched epithelial cell adherence and viability, as well as mechanical characteristics of different concentrations of GelMA. All concentrations of hydrogel show long-term biocompatibility for the mono- and co-cultures, despite the goblet-like cells (LS174T) showing lower performance than enterocyte-like cells (Caco-2). The mechanical strength of all hydrogel concentrations was in a physiologically relevant range to be used as scaffold material for intestinal cells. Lastly, we examined the effect of the two sterilization methods, ethylene oxide (EO) and 70% ethanol followed by UVC (EtOH/UVC). We found that the impact of the two methods on the mechanical characteristics was minimal, and we did not find a significant effect between the two methods on cell viability and confluency of Caco-2 cells seeded on the GelMA hydrogels. Based on these results, we conclude that GelMA is a suitable material as a scaffold for intestinal cell types in terms of permeability, mechanical strength and biocompatibility. These findings contribute to the growing field of in vitro modeling of the gut and moves the field further to ensuring more translatable research on host-microbe interactions.

The extracellular matrix (ECM) plays a crucial role in organoid cultures by supporting cell proliferation and differentiation. A key feature of the ECM is its mechanical influence on the surrounding cells, directly affecting their behavior. Matrigel, the most commonly used ECM, is limited by its animal-derived origin, batch variability, and uncontrollable mechanical properties, restricting its use in 3D cell-model-based mechanobiological studies. Poly(2-alkyl-2-oxazoline) (PAOx) synthetic hydrogels represent an appealing alternative because of their reproducibility and versatile chemistry, enabling tuning of hydrogel stiffness and functionalization. Here, we studied PAOx hydrogels with differing compressive moduli for their potential to support 3D cell growth. PAOx hydrogels support spheroid and organoid growth over several days without the addition of ECM components. Furthermore, we discovered intestinal organoid epithelial polarity reversion in PAOx hydrogels and demonstrate how the tunable mechanical properties of PAOx can be used to study effects on the morphology and oxygenation of live multicellular spheroids.

As an emerging technology, organoids are promising new tools for basic and translational research in disease. Currently, the culture of organoids relies mainly on a type of unknown composition scaffold, namely Matrigel, which may pose problems in studying the effect of mechanical properties on organoids. Hydrogels, a new material with adjustable mechanical properties, can adapt to current studies. In this review, we summarized the synthesis of recent advance in developing definite hydrogel scaffolds for organoid culture and identified the critical parameters for regulating mechanical properties. In addition, classified by different mechanical properties like stiffness and viscoelasticity, we concluded the effect of mechanical properties on the development of organoids and tumor organoids. We hope this review enhances the understanding of the development of organoids by hydrogels and provides more practical approaches to investigating them.

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

Organoids have revolutionized the whole field of biology with their ability to model complex three-dimensional human organs in vitro. Intestinal organoids were especially consequential as the first successful long-term culture of intestinal stem cells, which raised hopes for translational medical applications. Despite significant contributions to basic research, challenges remain to develop intestinal organoids into clinical tools for diagnosis, prognosis, and therapy. In this review, we outline the current state of translational research involving adult stem cell and pluripotent stem cell derived intestinal organoids, highlighting the advances and limitations in disease modeling, drug-screening, personalized medicine, and stem cell therapy. Preclinical studies have demonstrated a remarkable functional recapitulation of infectious and genetic diseases, and there is mounting evidence for the reliability of intestinal organoids as a patient-specific avatar. Breakthroughs now allow the generation of structurally and cellularly complex intestinal models to better capture a wider range of intestinal pathophysiology. As the field develops and evolves, there is a need for standardized frameworks for generation, culture, storage, and analysis of intestinal organoids to ensure reproducibility, comparability, and interpretability of these preclinical and clinical studies to ultimately enable clinical translation.

Two-dimensional (2D) nanoarchitectonics involve the creation of functional material assemblies and structures at the nanoscopic level by combining and organizing nanoscale components through various strategies, such as chemical and physical reforming, atomic and molecular manipulation, and self-assembly. Significant advancements have been made in the field, with the goal of producing functional materials from these nanoscale components. 2D nanomaterials, in particular, have gained substantial attention due to their large surface areas which are ideal for numerous surface-active applications. In this review article, nanoarchitectonics of 2D nanomaterials based biomedical applications are discussed. We aim to provide a concise overview of how nanoarchitectonics using 2D nanomaterials can be applied to dental healthcare, with an emphasis on biosensing and drug delivery. By offering a deeper understanding of nanoarchitectonics with programmable structures and predictable properties, we hope to inspire new innovations in the dental bioapplications of 2D nanomaterials.

Trichinella spiralis and porcine epidemic diarrhea virus (PEDV) are two infectious swine pathogens. Parasite excretory/secretory antigens play critical roles in various disease processes. To explore the coexistence mechanism of two pathogens infecting the same host, the intestinal organoid was utilized to reproduce these biological processes. In this study, we investigated the effects of T. spiralis excretory/secretory antigens (TsES) on PEDV-induced inflammatory regulation, lesion recovery, and mucosal barrier repair in porcine intestinal organoids. The results showed that PEDV effectively infected the porcine intestinal organoids. Next, TsES inhibited pro-inflammatory cytokines and increased the anti-inflammatory cytokines produced by PEDV-infected porcine intestinal organoids. Further, four-dimensional (4D) label-free quantitative proteomics and western blotting confirmed that TsES regulate the inflammation caused by PEDV infection through the nuclear factor kappa-B (NF-κB) pathway. In addition, TsES promoted cell proliferation, inhibited apoptosis, and reduced PEDV-induced lesions in intestinal organoids. The elevated secretory immunoglobulin A (sIgA) levels caused by PEDV infection were downregulated by TsES treatment in intestinal organoids. TsES treatment reversed the mucosal barrier damage caused by PEDV infection in intestinal organoids. Finally, PEDV replication increased after TsES treatment in organoids. We highlight the potential of TsES to ameliorate PEDV-induced inflammation, mucosal lesions, and barrier damage in porcine intestinal organoids. TsES also contribute to PEDV replication. This study presents a novel research model for research on host-virus-parasite interactions, while also providing a theoretical foundation to consider parasite derivatives as a potential adjunctive therapy for intestinal inflammation.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

Neurological injuries and diseases are a leading cause of disability worldwide, underscoring the urgent need for effective therapies. Neural regaining and enhancement therapies are seen as the most promising strategies for restoring neural function, offering hope for individuals affected by these conditions. Despite their promise, the path from animal research to clinical application is fraught with challenges. Neuroengineering, particularly through the use of biomaterials, has emerged as a key field that is paving the way for innovative solutions to these challenges. It seeks to understand and treat neurological disorders, unravel the nature of consciousness, and explore the mechanisms of memory and the brain's relationship with behavior, offering solutions for neural tissue engineering, neural interfaces and targeted drug delivery systems. These biomaterials, including both natural and synthetic types, are designed to replicate the cellular environment of the brain, thereby facilitating neural repair. This review aims to provide a comprehensive overview for biomaterials in neuroengineering, highlighting their application in neural functional regaining and enhancement across both basic research and clinical practice. It covers recent developments in biomaterial-based products, including 2D to 3D bioprinted scaffolds for cell and organoid culture, brain-on-a-chip systems, biomimetic electrodes and brain-computer interfaces. It also explores artificial synapses and neural networks, discussing their applications in modeling neural microenvironments for repair and regeneration, neural modulation and manipulation and the integration of traditional Chinese medicine. This review serves as a comprehensive guide to the role of biomaterials in advancing neuroengineering solutions, providing insights into the ongoing efforts to bridge the gap between innovation and clinical application.